Lipopolysaccharide Down-regulates AMPK/PGC-1? Signaling Pathway And Inhibits White Adipose Tissue Browning

Objective:The adenosine 5'-monophosphate-activated protein kinase(AMPK)/ peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC-1?)signaling pathway mediates white fat tissue(WAT)browning.Inducing WAT browning to increase energy expenditure is essential for the intervention of metabolic diseases.However,obesity makes this process insensitive,and the underlying mechanism is unclear.Studies have shown that "metabolic endotoxemia" is common in obese people.Therefore,this study aims to investigate whether endotoxin(lipopolysaccharide,LPS)can inhibit the browning of adipocytes by affecting the AMPK / PGC-1? signaling pathway,and provide a theoretical basis for clarifying the role and mechanism of LPS in metabolic diseases.Methods:1.Animal experiment Forty-eight 6-week-old C57 BL / 6 mice were divided into two groups: the standard mouse feed group(n=32)and the 45% kcal fat high-fat feed group(n=16).The weight of mice were monitored weekly.After 19 weeks,the standard mouse feed group was randomly divided into four groups(n=8): normal control group(ND),injection of CL316,243 group(CL),injection of LPS group(LPS),injection of LPS and CL316,243group(LPS + CL);The 45% kcal fat high-fat diet feed group was randomly divided into two groups(n=8): high-fat diet group(HFD),and high-fat diet injection CL316,243 group(HFD + CL).The food intake of mice were monitored twice a week.After the experiment was finished,mice were anesthetized with 1% sodium pentobarbital,and the inguinal and epididymal adipose tissues were separated and weighed.Adipose tissue was divided into two parts,one part was placed in sterile specimen tubes and quickly frozen at-80 ° C.Western blot detects the proteins expression of LBP,TLR4,AMPK,p-AMPK,PGC-1? and UCP1 and RT-q PCR detects UCP1 m RNA expression;one part was fixed in 4%paraformaldehyde for 24 h,and the morphological changes of fat cells were observed byHE stain.2.Experiment in vitro3T3-L1 preadipocytes were cultured in DMEM high glucose solution containing 10%FBS and 1% penicillin.The cells in the logarithmic growth phase were inoculated into a six-well plate,and the cells were induced to become adipocytes after the cells completely confluent.The morphology of the adipocytes was observed under a microscope and identified with oil red O staining.The mature adipocytes were divided into four groups:control group(NC),10 ?mol / L CL316,243 group(CL),10 ?mol / L CL316,243 combined with 15 ?mol / L AMPK inhibitor(CC)(added 2 h in advance)group(CC + CL),10 ?mol / L CL316,243 combined with 1 ?g / ml LPS(added 24 h in advance)group(LPS+ CL).After the intervention,western blot detects the protein expression of AMPK,p-AMPK,PGC-1?,UCP1 and RT-q PCR detects UCP1 m RNA expression and cellular immunofluorescence detects the change of mitochondrial content.3.Statistical analysis All date are presented as the means ± SEM.Statistical analyses were conducted by SPSS22.0 system and Graph Pad Prism 6(Version 6.02).Student's t test,one-way ANOVA or Mann-Whitney U were used for comparison between groups.P < 0.05 was regarded as statistically significant.Results:1.Compared with ND group,the weight of mice in group HFD began to increase at the third week,and the difference has statistically significant(P <0.05);After 19 weeks,the weight of the mice,inguinal white adipose tissue and epididymal white adipose tissue in group HFD were significantly increased(P <0.001).2.Compared with ND group,mice in group CL lower weight(P <0.05),and there was no significant difference in food intake(P >0.05);HE staining shown that the morphology of adipocytes changed from single-room lipids to multi-room lipids;the expressions of UCP1 protein(P <0.001)and m RNA(P <0.05)increased significantly.Compared with HFD group,there was no statistical difference in body weight and food intake in group HFD + CL(P >0.05);HE staining shown that the morphology of adipocytes did not change significantly.Compared with CL group,the expressions of UCP1 protein(P <0.001),UCP1 m RNA(P <0.05),PGC-1?(P <0.01)protein and AMPK protein phosphorylation level(P <0.001)were significant declined in group HFD + CL.3.Compared with ND group,the expressions of LBP and TLR4 protein in inguinal white adipose tissue of mice were increased in groups HFD + CL and LPS + CL(P<0.001);HE staining shown that the morphology of adipocytes did not change significantly in group LPS + CL.Compared with CL group,the expressions of UCP1protein(P <0.001),UCP1 m RNA(P <0.05),PGC-1?(P <0.01)protein and AMPK protein phosphorylation level(P <0.001)were significant declined in group LPS + CL.4.In the cell experiments,compared with NC group,the expressions of UCP1 protein(P <0.01),UCP1 m RNA(P <0.05),PGC-1? protein(P <0.01)and AMPK protein phosphorylation level(P <0.001)was significantly increased in group CL;the cellular immunofluorescence shown that the mitochondrial content was increased.Compared with CL group,the expressions of UCP1 protein(P <0.01),UCP1 m RNA(P <0.01),PGC-1?protein(P <0.001)and AMPK protein phosphorylation level(P <0.001)were significantly reduced in group CC + CL;the expressions of UCP1 protein(P <0.01),UCP1 m RNA(P<0.01),PGC-1?(P <0.01)and AMPK protein phosphorylation level(P <0.001)were also significantly reduced in the group LPS + CL.Conclusion:High fat diet-induced obesity mice inhibited i WAT browning mediated by CL316,243,which may be related to LPS down-regulation of AMPK / PGC-1? signaling pathway in adipocytes.